Phase Transitions in Chemically Fueled, Multiphase Complex Coacervate Droplets

Abstract Membraneless organelles are droplets in the cytosol that are regulated by chemical reactions. Increasing studies suggest that they are internally organized. However, how these subcompartments are regulated remains elusive. Herein, we describe a complex coacervate‐based model composed of two polyanions and a short peptide. With a chemical reaction cycle, we control the affinity of the peptide for the polyelectrolytes leading to distinct regimes inside the phase diagram. We study the transitions from one regime to another and identify new transitions that can only occur under kinetic control. Finally, we show that the chemical reaction cycle controls the liquidity of the droplets offering insights into how active processes inside cells play an important role in tuning the liquid state of membraneless organelles. Our work demonstrates that not only thermodynamic properties but also kinetics should be considered in the organization of multiple phases in droplets.


Isothermal titration calorimetry (ITC
UV/Vis Spectroscopy. The UV/Vis measurements were carried out using a Microplate Spectrophotometer (Thermo Scientific Multiskan GO). Samples (100 μL) were directly prepared into a 96 well-plate (tissue culture plate non-treated) and the absorbance as a measure for turbidity was monitored at 600 nm every minute. Measurements were performed at 25 °C. Each experiment was performed in triplicate.
Fluorescence Spectroscopy. Fluorescence spectroscopy was performed on a Jasco (Jasco FP-8300) spectrofluorimeter. Samples (100 µL) were added to a cuvette and the fluorescence was measured at specific wavelengths depending on the dye (see confocal settings). Measurements were performed at 24°C. Each experiment was performed in triplicate.

Confocal Fluorescence Microscopy.
Confocal fluorescence microscopy was performed on a Leica TCS SP8 confocal microscope using a 63x water immersion objective (1.2 NA). Samples (V = 30 µL) were prepared as described above in PVA-coated [2] micro-well plates (ibidi, µ-Slide Angiogenesis Glass Bottom), but with 0.15 µM Cy3-RNA, 0.15 µM Cy5-RNA, 0.15 µM Cy5-pSS and/or 1 µM NBD-peptide as the fluorescent dye. Samples were excited with 488 nm (NBD), 552 nm (Cy3), and 638 nm (Cy5) and imaged at 498-550 nm, 560-630 nm, and 650-710 nm, respectively. A sequential scan was employed when more than one dye was present in the sample. Images of passive droplets were typically obtained 10 minutes after droplet formation. Measurements were performed at 24 °C. Fluorescent recovery after photobleaching (FRAP). The diffusivity of the molecules inside active droplets was measured via spot bleaching. The region of interest (ROI) was set to a radius of 0.8 µm inside droplets with a diameter of a couple of micrometers. For peptide 2, samples containing 1 µM NBD-GRGRGRGD-OH (fluorescent peptide 2) at standard conditions were bleached and excited with a 488 nm laser and imaged at 498-550 nm with an image size of 144x80 pixels. 5 pre-bleaches (every 53 ms) were acquired followed by 3 bleaching pulses (every 53 ms). After that, fluorescence recovery was tracked every 53-500 milliseconds with a total of up to 107 images. For pSS, samples containing 0.15 µM Cy5-pSS at standard conditions were bleached and exited at 638 nm and imaged at 650-710 nm with an image size of 256x100 pixels. 5 pre-bleaches (every 0.19 s) were acquired followed by 10 bleaching pulses (every 0.19 s). After that, fluorescence recovery was tracked every 0.19-1.0 seconds with a total of up to 163 images. To measure the diffusivity of pSS and pU in samples with either 10 mM pSS or pU at standard conditions with 0.15 µM dye, samples were bleached and exited at either 552 nm or 638 nm and imaged at 560-630 or 650-710 nm with an image size of 512x64 pixels. The raw data was then grouped into time regimes after fuel addition (e.g. 0-7 min, 10-15 min,..). After double normalization [3] , the data were fitted to a first-order exponential equation [4] with the spot size and the diffusion coefficient to obtain the diffusion coefficient and half-time recovery, as previously described. [2] For the vacuole phase, it was only possible to immobilize the vacuole for a couple of seconds. As a result, we could only accurately determine the diffusion of the peptide in the vacuole since it is a small molecule, and its fluorescent intensity recovers much faster than pU and pSS. Here, the image size was 80x40 pixels and the raw data was only subtracted from the background without normalization of neighboring droplets. To measure the diffusivity of pU in the pSS phase of active multiphase droplets, we hybridized pU with Cy5-A15-pU in presence of sulforhodamine (to better visualize the droplets). Samples were then bleached and excited with a 638 nm laser and imaged at 650-710 nm with an image size of 144x80 pixels. 5 pre-bleaches (every 53 ms) were acquired followed by 10 bleaching pulses (every 53 ms). After that, fluorescence recovery was tracked every 53-500 milliseconds with a total of up to 107 images. Since the partitioning of pU in the pSS phase is close to 1, we did not subtract the background but only corrected it for photobleaching by a neighboring droplet. Error bars for all experiments show the standard deviation from two experiments with three droplets each (N=6) per time point.

Microfluidic chip production:
Microfluidic PDMS (Polydimethylsiloxane, Sylgard 184, Dow Corining)-based devices were designed with QCAD-pro (RibbonSoft GmbH) and fabricated using photo-and soft-lithography [5] as previously described. [6] Microfluidic droplet formation: Surfactant-stabilized water in oil droplets was produced using 2% 008-FluoroSurfactant in 3M Novec7500 as the oil phase, fuel in MQ water as one of the water phases and peptide 2, polyanions and dyes in MES buffer at pH 5.3 as the second water phase. Both water phases contained the doubled concentrations of the respective components with respect to the standard conditions and were mixed in a 1-to-1 ratio to yield the standard concentrations inside the microfluidic droplets. Both aqueous phases and the oil phase were injected into the microfluidic PDMS-based device through polytetrafluoroethylene (PTFE) tubes (0.4-0.9 mm, Bola, Germany) using a flow control system (ELVEFLOW Pressure Controller OB1 MK3). Typically, pressures of 610 mbar for the water phases and 810 mbar for the oil phase were used to produce stable water in oil droplets with a diameter of 40 µm.
HPLC. The concentration profiles of the fuel, peptide 2, and activated peptide 2 during the chemical reaction cycle were monitored over time using analytical HPLC (ThermoFisher, Vanquish Duo UHPLC, HPLC) with a Hypersil Gold 100 2.1 mm C18 column (3 mm pore size).
To determine the EDC concentration over time, a turbid sample (V = 10 µL) of active droplets in an HPLC vial with an inlet at standard conditions was diluted with 10 µL of an aqueous NaCl solution (4M) at a given time point and directly injected into the HPLC. Separation was performed using a linear gradient of ACN (2 to 98%) and water with 0.1% TFA and the chromatogram was analyzed using detectors at 220 nm and 254 nm. To determine the activated peptide 2 concentration, a benzylamine quench was used. [1b, 7] Briefly, at each time point, 10 µL from a sample containing active droplets (V = 120 µL) in a 96 well plate were added into 20 µL of an aqueous solution of benzylamine (300 mM) in an HPLC vial. After the addition of 10 µL of an aqueous NaCl solution (4M), the samples were directly injected into the HPLC with the settings from above. All measurements were performed at 25 °C. Each experiment was performed in triplicate.
Kinetic model. A kinetic model written in MATLAB was used to predict the evolution of fuel, peptide 2, and activated peptide 2 over time. The model is described in detail in our previous work.
Measuring the concentrations in the phase outside of the droplets (coutside). Passive droplets: a solution of passive droplets (V = 150 µL) prepared at standard conditions with 1 µM NBD-GRGRGRGN-NH2 (fluorescent peptide 1) and 0.15 µM Cy5-pSS in an Eppendorf tube was vortexed for 10 seconds and then centrifuged for 10 minutes at 20,412×g. The supernatant (V = 100 µL) was removed and added to an Eppendorf tube containing 1 µL of an aqueous solution of NaCl (4 M) to dissolve residual turbidity. The fluorescence of the sample was then measured on the fluorimeter. To account for the dependence of the fluorescence of dyes on their environment, [8] we prepared an identical sample w/o dye and added the supernatant (85-95 µL) to an Eppendorf tube containing 1 µL of an aqueous solution of NaCl (4 M). To the clear solution, we then added the same amount of dye (5-15 µL) as in the previous sample and measured its fluorescence intensity. The intensity ratio between these 2 samples was therefore the fraction of the fluorescent molecules that remained in the supernatant. Error bars show the standard deviation from the average (N=3). Active droplets: to a solution of active droplets (V = 150 µL) prepared at standard conditions with 1 µM NBD-GRGRGRGD-OH (fluorescent peptide 2), 0.15 µM Cy3-A15 or 0.15 µM Cy5-pSS, 50 mM EDC was added. At a given timepoint, the sample was centrifuged for 1 min at 20,412×g. The fraction of fluorescent molecules that remained in the supernatant was then measured and quantified as for passive droplets. Since fluorescent peptide 2 can also be activated by the fuel, the measured intensity is reflecting the partitioning of peptide 2 and activated peptide 2 combined. Error bars show the standard deviation from the average (N=3).

Calculating the concentrations inside the droplets and the resulting partitioning coefficient.
We determined the droplet volumes of passive droplets at standard conditions after centrifugation of the turbid suspensions (V = 150 µL) for 10 minutes at 20,412×g. The resulting droplet pellet (0.2 -1.5 µL) was then compared to size standards visually. We then calculated the concentration in the droplet phase by subtracting the number of molecules in the phase outside of the droplets from the total amount in the solution. Partitioning coefficients K were obtained by the ratio of the concentration in the droplets and the concentration outside of the droplets.
Calculating concentrations inside the vacuole phase. We measured the fluorescence intensity profiles of active droplets via confocal fluorescence microscopy, 22 minutes after the addition of 50 mM fuel under standard conditions. The resulting partitioning coefficients Kvacuole were then multiplied with the concentration in the phase outside of the droplets coutside obtained from the fluorimeter (see method above) to give the concentrations inside the vacuole cvacuole (Table S2) , where D is the diffusion coefficient, kB is the Boltzmann constant, T is the temperature, η is the viscosity and Rh is the hydrodynamic radius. Under the assumption of the droplets behaving as equilibrium Newtonian liquids, the droplets' viscosity can be estimated as a first approximation.
[9] D values of pSS were used from FRAP experiments. Rh of pSS was assumed to be approximately 10 nm based on the literature. [10] Determining the critical coacervation concentration (CCC) and critical salt concentration (CSC) of passive droplets. The CCC concentrations of passive droplets were determined by increasing the amount of peptide 1 gradually by pipetting increasing amounts of a 100 mM stock solution into a sample containing either 10 mM pSS or 10 mM pU in 200 mM MES at pH 5.3. Turbidity was measured by UV/vis spectrometry at 600 nm. Turbidity values above the blank value (0.085 absorbance) were taken as the indicator for phase separation. Critical salt concentrations were determined accordingly, but different amounts of a 4M NaCl stock solution were added to samples containing 2.5 mM of peptide 1, 10 mM of pSS or 10 mM pU in 200 mM MES at pH 5.3.

Supplementary discussion 1:
We performed a control experiment to indirectly demonstrate that A15 must be mostly hybridized even in the droplet environment. For that, we used our conditions for active multiphase droplets based on pU and pSS. As dyes, we used Cy3-A15-pU and Cy5-U15 instead of Cy5-pSS. Cy5-U15 cannot base-pair with pU. Indeed, we observed that U15 localized into the pSS phase during vacuole formation ( Figure S7). Thus, there is a significant difference in the partitioning of non-hybridized U15 compared to hybridized A15. A15 and U15 should only show disparate partitioning in multiphase droplets if base-pairing is present, [11] i.e., the pU-phase selects for one of the two nucleic acids via hybridization. Thus, we conclude that the Cy3emission from A15 indicates pU's location.

Supplementary discussion 2:
The pU shell in active multiphase droplets was too thin for FRAP spot bleaching experiments. Instead, we performed FRAP on pU-only droplets (10 mM) as an indirect measure for the pU shell ( Figure S13). To verify whether this assumption was true, we performed control experiments on pSS-only droplets ( Figure S15). Here, the diffusivities obtained for the peptide and pSS were in the same range as for the pSS core in multiphase droplets, indicating that the measured diffusivities in pU-only droplets can be used as an estimate for the pU shell in multiphase droplets.